A unique bacterial secretion machinery with multiple secretion centers.

The Porphyromonas gingivalis type IX secretion system (T9SS) promotes periodontal disease by secreting gingipains and other virulence factors. By in situ cryoelectron tomography, we report that the P. gingivalis T9SS consists of 18 PorM dimers arranged as a large, caged ring in the periplasm. Near the outer membrane, PorM dimers interact with a PorKN ring complex of ∼52 nm in diameter. PorMKN translocation complexes of a given T9SS adopt distinct conformations energized by the proton motive force, suggestive of different activation states. At the inner membrane, PorM associates with a cytoplasmic complex that exhibits 12-fold symmetry and requires both PorM and PorL for assembly. Activated motors deliver substrates across the outer membrane via one of eight Sov translocons arranged in a ring. The T9SSs are unique among known secretion systems in bacteria and eukaryotes in their assembly as supramolecular machines composed of apparently independently functioning translocation motors and export pores.

The short-term efficacy of neoadjuvant SOX versus SOX plus immune checkpoint inhibitor following laparoscopic gastrectomy for locally advanced gastric cancer: a multicenter retrospective cohort study in China.

BACKGROUND: This study aims to evaluate the short-term efficacy for locally advanced gastric cancer (LAGC) who accepted laparoscopic gastrectomy (LG) after neoadjuvant SOX versus SOX plus immune checkpoint inhibitors (ICIs). METHODS: LAGC patients who accepted LG after neoadjuvant SOX (SOX-LG, n = 169) and SOX plus ICIs (SOX + ICIs-LG, n = 140) in three medical centers between Jan 2020 and Mar 2024 were analyzed. We compared the tumor regression, treatment-related adverse events (TRAEs), perioperative safety between two groups, and explored the risk factors of postoperative complications (POCs) for LG after neoadjuvant therapy. RESULTS: The baseline characteristics were comparable between two groups (P > 0.05). SOX + ICIs-LG group acquired a higher proportion of objective response (63.6% vs. 46.7%, P = 0.003), major pathological response (43.6% vs. 31.4%, P = 0.001), and pathological complete response (17.9% vs. 9.5%, P = 0.030). There were no significant differences in the TRAEs rates, operation time, R0 resection, retrieved lymph nodes, postoperative first flatus, and hospitalized days, overall and severe POCs between two groups (P > 0.05). Patients in the SOX-ICIs-LG group had lower estimated blood loss (EBL) compared with SOX-LG (P = 0.001). Multivariate analysis showed that more EBL (P = 0.003) and prognostic nutritional index (PNI) < 40 (P = 0.005) were independent risk factors of POCs for LG after neoadjuvant therapy. CONCLUSION: Neoadjuvant SOX plus ICIs brings better tumor regression and similar TRAEs compared with SOX alone for LAGC. SOX + ICIs-LG is safe and feasible to conduct with less EBL. Surgeons should focus on the perioperative management to control POCs for patients with PNI < 40 and more EBL.

Nebulized inhalation of plasma-activated water in the treatment of progressive moderate COVID-19 patients with antiviral treatment failure: a randomized controlled pilot trial.

BACKGROUND: Antiviral drugs show significant efficacy in non-severe COVID-19 cases, yet there remains a subset of moderate COVID-19 patients whose pneumonia continues to progress post a complete course of treatment. Plasma-activated water (PAW) possesses anti-SARS-CoV-2 properties. To explore the potential of PAW in improving pneumonia in COVID-19 patients following antiviral treatment failure, we conducted this study. METHODS: This was a randomized, controlled trial. Moderate COVID-19 patients with antiviral treatment failure were randomly assigned to the experimental group or the control group. They inhaled nebulized PAW or saline respectively. This was done twice daily for four consecutive days. We assessed improvement in chest CT on day 5, the rate of symptom resolution within 10 days, and safety. RESULTS: A total of 23 participants were included, with 11 receiving PAW and 12 receiving saline. The baseline characteristics of both groups were comparable. The experimental group showed a higher improvement rate in chest CT on day 5 (81.8% vs. 33.3%, p = 0.036). The cumulative disappearance rate of cough within 10 days was higher in the experimental group. Within 28 days, 4 patients in each group progressed to severe illness, and no patients died. No adverse reactions were reported from inhaling nebulized PAW. CONCLUSION: This pilot trial preliminarily confirmed that nebulized inhalation of PAW can alleviate pneumonia in moderate COVID-19 patients with antiviral treatment failure, with no adverse reactions observed. This still needs to be verified by large-scale studies. TRIAL REGISTRATION: Chinese Clinical Trial Registry; No.: ChiCTR2300078706 (retrospectively registered, 12/15/2023); URL: www.chictr.org.cn .

CLCA1 exacerbates lung inflammation via p38 MAPK pathway in acute respiratory distress syndrome.

Recent research has revealed that airway epithelial calcium-activated chloride channel-1 (CLCA1) is implicated in the inflammation of multiple human respiratory diseases, but the specific role in acute respiratory distress syndrome (ARDS) remains unknown. To investigate the role of CLCA1 in ARDS, 80 participants, including 26 ARDS patients, 26 patients with community-acquired pneumonia (CAP) and 28 control subjects, were enrolled in this study. As the result shows, the level of CLCA1 was significantly increased in ARDS patients and positively correlated with neutrophil infiltration and the poor prognosis of ARDS. Then, the level of CLCA1 also elevated in the LPS-induced ARDS mouse model, and the administration of CLCA1 significantly regulated the phenotypes of ARDS in mice, such as lung injury score, BALF protein concentration, neutrophils infiltration and the secretions of inflammatory factors. Furthermore, administration of CLCA1 substantially altered the phosphorylation of p38 in the ARDS mouse model, whereas repressing the expression of CLCA1 or inhibiting the activation of p38 both alleviated the inflammatory response of ARDS. In summary, CLCA1 was notably correlated with ARDS and exacerbated the ARDS phenotypes through the p38 MAPK pathway.

Contributions of F-specific subunits to the F plasmid-encoded type IV secretion system and F pilus.

F plasmids circulate widely among the Enterobacteriaceae through encoded type IV secretion systems (T4SSF s). Assembly of T4SSF s and associated F pili requires 10 VirB/VirD4-like Tra subunits and eight or more F-specific subunits. Recently, we presented evidence using in situ cryoelectron tomography (cryoET) that T4SSF s undergo structural transitions when activated for pilus production, and that assembled pili are deposited onto alternative basal platforms at the cell surface. Here, we deleted eight conserved F-specific genes from the MOBF12C plasmid pED208 and quantitated effects on plasmid transfer, pilus production by fluorescence microscopy, and elaboration of T4SSF structures by in situ cryoET. Mutant phenotypes supported the assignment of F-specific subunits into three functional Classes: (i) TraF, TraH, and TraW are required for all T4SSF -associated activities, (ii) TraU, TraN, and TrbC are nonessential but contribute significantly to distinct T4SSF functions, and (iii) TrbB is essential for F pilus production but not for plasmid transfer. Equivalent mutations in a phylogenetically distantly related MOB12A F plasmid conferred similar phenotypes and generally supported these Class assignments. We present a new structure-driven model in which F-specific subunits contribute to distinct steps of T4SSF assembly or activation to regulate DNA transfer and F pilus dynamics and deposition onto alternative platforms.

Predictors and outcomes of recurrent retroperitoneal liposarcoma: new insights into its recurrence patterns.

BACKGROUND: The clinical profiles of recurrent retroperitoneal liposarcoma (RLS) need to be explored. The recurrence patterns of RLS are controversial and ambiguous. METHODS: A total of 138 patients with recurrent RLS were finally recruited in the study. The analysis of overall survival (OS) and recurrence-free survival (RFS) was performed by Kaplan‒Meier analysis. To identify independent prognostic factors, all significant variables on univariate Cox regression analysis (P ≤ 0.05) were subjected to multivariate Cox regression analysis. The corresponding nomogram model was further built to predict the survival status of patients. RESULTS: Among patients, the 1-, 3-, and 5-year OS rates were 70.7%, 35.9% and 30.9%, respectively. The 1-, 3- and 5-year RFS rates of the 55 patients who underwent R0 resection were 76.1%, 50.8% and 34.4%, respectively. The multivariate analysis revealed that resection method, tumor size, status of pathological differentiation, pathological subtypes and recurrence pattern were independent risk factors for OS or RFS. Patients with distant recurrence (DR) pattern usually had multifocal tumors (90.5% vs. 74.7%, P < 0.05); they were prone to experience changes of pathological differentiation (69.9% vs. 33.3%, P < 0.05) and had a better prognosis than those with local recurrence (LR) pattern. R0 resection and combined organ resection favored the survival of patients with DR pattern in some cases. CONCLUSIONS: Patients with DR pattern had better prognosis, and they may benefit more from aggressive combined resection than those with LR pattern. Classifying the recurrence patterns of RLS provides guidance for individualized clinical management of recurrent RLS.

Five-year long-term comparison of robotic and laparoscopic gastrectomy for gastric cancer: a large single-center cohort study.

BACKGROUND: Robotic gastrectomy (RG) has been reported to be technically feasible and safe for patients with gastric cancer. However, 5-year long-term survival and recurrence outcomes for advanced gastric cancer have rarely been reported. This study aimed to compare the long-term oncologic outcomes between RG and laparoscopic gastrectomy (LG) for gastric cancer. METHODS: The general clinicopathological data of 1905 consecutive patients who underwent RG and LG were retrospectively collected at the Chinese People's Liberation Army General Hospital between November 2011 and October 2017. Propensity score matching (PSM) was used to match groups. The primary endpoints were 5-year disease-free survival (DFS) and overall survival (OS). RESULTS: After PSM, a well-balanced cohort of 283 patients in the RG group and 701 patients in the LG group were included in the analysis. The 5-year cumulative DFS rates were 67.28% in the robotic group and 70.41% in the laparoscopic group. The 5-year OS rate was 69.01% in the robotic group and 69.58% in the laparoscopic group. No significant differences in Kaplan-Meier survival curves for DFS (HR = 1.08, 95% CI 0.83-1.39, Log-rank P = 0.557) and OS (HR = 1.02, 95% CI 0.78-1.34, Log-rank P = 0.850) were observed between the 2 groups. In the subgroup analyses for potential confounding variables, there were no significant differences in 5-year DFS and 5-year OS survival between the 2 groups (P > 0.05), except for patients with pathological stage III and pathological stage N3 (P < 0.05). CONCLUSION: For patients with early gastric cancer, robotic and laparoscopic approaches have similar long-term survival. For patients with advanced gastric cancer, further studies need to be conducted to assess the long-term survival outcomes of RG.

Myeloid-specific blockade of Notch signaling alleviates murine pulmonary fibrosis through regulating monocyte-derived Ly6clo MHCIIhi alveolar macrophages recruitment and TGF-ß secretion.

Macrophages in lung, including resident alveolar macrophages (AMs) and interstitial macrophages (IMs), and monocyte-derived macrophages, play important roles in pulmonary fibrosis (PF), but mechanisms underlying their differential regulation remain unclear. Recombination signal-binding protein Jκ (RBP-J)-mediated Notch signaling regulates macrophage development and phenotype. Here, using bleomycin-induced fibrosis model combined with myeloid-specific RBP-J disruption (RBP-JcKO ) mouse, we investigated the role of Notch signaling in macrophages during PF. Compared with the control, RBP-JcKO mice exhibited alleviated lung fibrosis as manifested by reduced collagen deposition and inflammation, and decreased TGF-ß production. FACS analysis suggested that decreased Ly6clo MHCIIhi AMs might make the major contribution to attenuated fibrogenesis in RBP-JcKO mice, probably by reduced inflammatory factor release and enhanced matrix metalloproteinases expression. Using clodronate-mediated macrophage depletion in RBP-JckO mice, we demonstrated that embryonic-derived AMs play negligible role in lung fibrosis, which was further supported by adoptive transfer experiments. Moreover, on CCR2 knockout background, the effect of RBP-J deficiency on fibrogenesis was not elicited, suggesting that Notch regulated monocyte-derived AMs. Co-culture experiment showed that monocyte-derived AMs from RBP-JcKO mice exhibit reduced myofibroblast activation due to decreased TGF-ß secretion. In conclusion, monocyte-derived Ly6clo MHCIIhi AMs, which are regulated by RBP-J-mediated Notch signaling, play an essential role in lung fibrosis.

Mechanistic insights into staphylopine-mediated metal acquisition.

Metal acquisition is vital to pathogens for successful infection within hosts. Staphylopine (StP), a broad-spectrum metallophore biosynthesized by the major human pathogen, Staphylococcus aureus, plays a central role in transition-metal acquisition and bacterial virulence. The StP-like biosynthesis loci are present in various pathogens, and the proteins responsible for StP/metal transportation have been determined. However, the molecular mechanisms of how StP/metal complexes are recognized and transported remain unknown. We report multiple structures of the extracytoplasmic solute-binding protein CntA from the StP/metal transportation system in apo form and in complex with StP and three different metals. We elucidated a sophisticated metal-bound StP recognition mechanism and determined that StP/metal binding triggers a notable interdomain conformational change in CntA. Furthermore, CRISPR/Cas9-mediated single-base substitution mutations and biochemical analysis highlight the importance of StP/metal recognition for StP/metal acquisition. These discoveries provide critical insights into the study of novel metal-acquisition mechanisms in microbes.

Molecular Cytogenetic Analysis of the Introgression between Agropyron cristatum P Genome and Wheat Genome.

Agropyron cristatum (2n = 4x = 28, PPPP) is an important wild relative of common wheat (Triticum aestivum L., 2n = 6x = 42). A previous report showed that the wheat-A. cristatum 6P translocation line WAT655 carrying A. cristatum 6PS (0.81-1.00) exhibited high resistance to prevalent physiological races of stripe rust (CYR32 and CYR33). In this study, three disease resistance-related transcripts, which were mapped to A. cristatum 6PS (0.81-1.00) through the analysis of specific molecular markers, were acquired from among A. cristatum full-length transcripts. The BC5F2 and BC5F2:3 genetic populations of the translocation line WAT655 were analyzed by using three disease resistance-related gene markers, A. cristatum P genome-specific markers, and fluorescence in situ hybridization (FISH). The results revealed that the introgression between A. cristatum P genome and wheat genome was observed in progenies of the genetic populations of the translocation line WAT655 and the physical positions of the three genes were considerably adjacent on A. cristatum 6PS (0.81-1.00) according to the FISH results. Additionally, kompetitive allele-specific PCR (KASP) markers of the three genes were developed to detect and acquire 24 breeding lines selected from the progenies of the distant hybridization of wheat and A. cristatum, which showed resistance to physiological races of stripe rust (CYR32 and CYR33) and other desirable agronomic traits according to the field investigation. In conclusion, this study not only provides new insights into the introgression between A. cristatum P genome and wheat genome but also provides the desirable germplasms for breeding practice.

Alternative Splicing of TaGS3 Differentially Regulates Grain Weight and Size in Bread Wheat.

The heterotrimeric G-protein mediates growth and development by perceiving and transmitting signals in multiple organisms. Alternative splicing (AS), a vital process for regulating gene expression at the post-transcriptional level, plays a significant role in plant adaptation and evolution. Here, we identified five splicing variants of Gγ subunit gene TaGS3 (TaGS3.1 to TaGS3.5), which showed expression divergence during wheat polyploidization, and differential function in grain weight and size determination. TaGS3.1 overexpression significantly reduced grain weight by 5.89% and grain length by 5.04%, while TaGS3.2-3.4 overexpression did not significantly alter grain size compared to wild type. Overexpressing TaGS3.5 significantly increased the grain weight by 5.70% and grain length by 4.30%. Biochemical assays revealed that TaGS3 isoforms (TaGS3.1-3.4) with an intact OSR domain interact with WGB1 to form active Gßγ heterodimers that further interact with WGA1 to form inactive Gαßγ heterotrimers. Truncated isoforms TaGS3.2-3.4 , which lack the C-terminal Cys-rich region but have enhanced binding affinity to WGB1, antagonistically compete with TaGS3.1 to bind WGB1, while TaGS3.5 with an incomplete OSR domain does not interact with WGB1. Taking these observations together, we proposed that TaGS3 differentially regulates grain size via AS, providing a strategy by which the grain size is fine-tuned and regulated at the post-transcriptional level.

Risk factors for hospital-acquired influenza A and patient characteristics: a matched case-control study.

BACKGROUND: While hospital-acquired influenza A results in an additional cost burden and considerable mortality in patients, its risk factors are unknown. We aimed to describe the characteristics of patients vulnerable to hospital-acquired influenza A and to identify its risk factors to assist clinicians control hospital-acquired infections and reduce the burden of treatment. METHODS: A case-control study was conducted among hospitalized patients aged ≥18 years at a tertiary level teaching hospital during the 2018-2019 influenza A season. Patient data were retrieved from hospital-based electronic medical records. Hospital-acquired influenza A was defined as a case of influenza A diagnosed 7 days or more after admission, in a patient with no evidence of influenza A infection on admission. The controls without influenza A were selected among patients exposed to the same setting and time period. We identified risk factors using conditional logistic regression and described the characteristics of hospital-acquired influenza A by comparing the clinical data of infected patients and the controls. RESULTS: Of the 412 hospitalized patients with influenza A from all the departments in the study hospital, 93 (22.6%) cases were classified as hospital-acquired. The most common comorbidities of the 93 cases were hypertension (41.9%), coronary heart disease (21.5%), and cerebrovascular disease (20.4%). Before the onset of hospital-acquired influenza A, patients presented more lymphocytopenia (51.6% vs 35.5%, P = 0.027), hypoalbuminemia (78.5% vs 57.0%, P = 0.002), and pleural effusion (26.9% vs 9.7%, P = 0.002) than the matched controls. Infected patients also had longer hospital stays (18 days vs 14 days, P = 0.002), and higher mortality rates (10.8% vs 2.2%, P = 0.017) than the matched controls. Lymphocytopenia (odds ratio [OR]: 3.11; 95% confidence interval [CI]: 1.24-7.80; P = 0.016), hypoalbuminemia (OR: 2.24; 95% CI: 1.10-4.57; P = 0.027), and pleural effusion (OR: 3.09; 95% CI: 1.26-7.58; P = 0.014) were independently associated with hospital-acquired influenza A. CONCLUSIONS: Lymphocytopenia, hypoalbuminemia and pleural effusion are independent risk factors that can help identify patients at high risk of hospital-acquired influenza A, which can extend hospital stay and is associated with a high mortality.

Molecular Cytogenetic Identification of Wheat-Aegilops Biuncialis 5Mb Disomic Addition Line with Tenacious and Black Glumes.

Production of wheat-alien disomic addition lines is of great value to the exploitation and utilization of elite genes originated from related species to wheat. In this study, a novel wheat-Aegilops biuncialis 5Mb disomic addition line WA317 was characterized by in situ hybridization (ISH) and specific-locus amplified fragment sequencing (SLAF-seq) markers. Compared to its parent Chinese Spring (CS), the glumes of WA317 had black color and were difficult to remove after harvesting, suggesting chromosome 5Mb carried gene(s) related to glume development and Triticeae domestication process. A total of 242 Ae. biuncialis SLAF-based markers (298 amplified patterns) were developed and further divided into four categories by Ae. biuncialis Y17, Ae. umbellulata Y139 and Ae. comosa Y258, including 172 markers amplifying the same bands of U and M genome, six and 102 markers amplifying U-specific and M-specific bands, respectively and eighteen markers amplifying specific bands in Y17. Among them, 45 markers had the specific amplifications in WA317 and were 5Mb specific markers. Taken together, line WA317 with tenacious and black glumes should serve as the foundation for understanding of the Triticeae domestication process and further exploitation of primitive alleles for wheat improvement. Ae. biuncialis SLAF-based markers can be used for studying syntenic relationships between U and M genomes as well as rapid tracking of U and M chromosomal segments in wheat background.

Deletion mapping and verification of an enhanced-grain number per spike locus from the 6PL chromosome arm of Agropyron cristatum in common wheat.

KEY MESSAGE: An enhanced-grain number per spike locus from Agropyron cristatum 6PL was mapped onto 6PL (0.27-0.51) via deletion mapping, and its effect was further verified by evaluating a newly created translocation line. Agropyron cristatum (2n = 4x = 28, PPPP) is an important wild relative of common wheat and carries many desirable yield-related traits. The wheat-A. cristatum 6P disomic addition line 4844-12 exhibited high grain number per spike (GNS), high spikelet number per spike (SNS), and high kernel number per spikelet (KNS). In this study, five A. cristatum 6P deletion lines, five wheat-A. cristatum 6P translocation lines, and genetic populations of these lines were used to map the enhanced-GNS locus from A. cristatum chromosome 6P, which were genotyped via genomic in situ hybridization, fluorescence in situ hybridization, or molecular markers. According to the evaluation of the agronomic traits in four growing seasons (2014-2015, 2015-2016, 2016-2017, and 2017-2018), we found that the deletion lines and the translocation lines carrying the long arm of A. cristatum chromosome 6P (6PL) exhibited high GNS, SNS, and KNS, and the enhanced-GNS locus was ultimately mapped onto 6PL (0.27-0.51). To verify the localization results, we created a new translocation line WAT650a (T5BL•5BS-6PL) that carried 6PL (0.35-0.42); this line exhibited higher GNS and SNS than the recipient parent Fukuhokomugi (Fukuho). Collectively, the enhanced-GNS locus of A. cristatum 6PL can be important for improving yield traits in common wheat; the translocation lines with the enhanced-GNS locus can serve as novel and valuable germplasm resources for wheat breeding.

A multicenter RCT of noninvasive ventilation in pneumonia-induced early mild acute respiratory distress syndrome.

RATIONALE: Our pilot study suggested that noninvasive ventilation (NIV) reduced the need for intubation compared with conventional administration of oxygen on patients with "early" stage of mild acute respiratory distress syndrome (ARDS, PaO2/FIO2 between 200 and 300). OBJECTIVES: To evaluate whether early NIV can reduce the need for invasive ventilation in patients with pneumonia-induced early mild ARDS. METHODS: Prospective, multicenter, randomized controlled trial (RCT) of NIV compared with conventional administration of oxygen through a Venturi mask. Primary outcome included the numbers of patients who met the intubation criteria. RESULTS: Two hundred subjects were randomized to NIV (n = 102) or control (n = 98) groups from 21 centers. Baseline characteristics were similar in the two groups. In the NIV group, PaO2/FIO2 became significantly higher than in the control group at 2 h after randomization and remained stable for the first 72 h. NIV did not decrease the proportion of patients requiring intubation than in the control group (11/102 vs. 9/98, 10.8% vs. 9.2%, p = 0.706). The ICU mortality was similar in the two groups (7/102 vs. 7/98, 4.9% vs. 3.1%, p = 0.721). Multivariate analysis showed minute ventilation greater than 11 L/min at 48 h was the independent risk factor for NIV failure (OR, 1.176 [95% CI, 1.005-1.379], p = 0.043). CONCLUSIONS: Treatment with NIV did not reduce the need for intubation among patients with pneumonia-induced early mild ARDS, despite the improved PaO2/FIO2 observed with NIV compared with standard oxygen therapy. High minute ventilation may predict NIV failure. TRIAL REGISTRATION: NCT01581229 . Registered 19 April 2012.

Challenges to and opportunities for the implementation of non-invasive positive pressure ventilation in the Asia-Pacific region.

Non-invasive positive pressure ventilation (NPPV) is undoubtedly one of the most significant advancements in mechanical ventilation technology in the past 30 years. With accumulating evidence from clinical studies and support from clinical guidelines, NPPV is now widely used in hospitals and increasingly prescribed for home therapy in the Asia-Pacific region. However, in comparison with the developed Western countries, overall use of NPPV in the region is lagging behind. This study reviews this imbalance of NPPV use both in the acute and domiciliary settings in the Asia-Pacific region. Important issues related to NPPV use are also discussed along with speculation around potential strategies that could promote wider implementation of NPPV in the region. We hope this review will stimulate interest in the clinical application and potential research avenues for NPPV in the Asia-Pacific region, and promote education and staff training in the technique.

CRISPR-Cas9 and CRISPR-Assisted Cytidine Deaminase Enable Precise and Efficient Genome Editing in Klebsiella pneumoniae.

Klebsiella pneumoniae is a promising industrial microorganism as well as a major human pathogen. The recent emergence of carbapenem-resistant K. pneumoniae has posed a serious threat to public health worldwide, emphasizing a dire need for novel therapeutic means against drug-resistant K. pneumoniae Despite the critical importance of genetics in bioengineering, physiology studies, and therapeutic-means development, genome editing, in particular, the highly desirable scarless genetic manipulation in K. pneumoniae, is often time-consuming and laborious. Here, we report a two-plasmid system, pCasKP-pSGKP, used for precise and iterative genome editing in K. pneumoniae By harnessing the clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9 genome cleavage system and the lambda Red recombination system, pCasKP-pSGKP enabled highly efficient genome editing in K. pneumoniae using a short repair template. Moreover, we developed a cytidine base-editing system, pBECKP, for precise C→T conversion in both the chromosomal and plasmid-borne genes by engineering the fusion of the cytidine deaminase APOBEC1 and a Cas9 nickase. By using both the pCasKP-pSGKP and the pBECKP tools, the blaKPC-2 gene was confirmed to be the major factor that contributed to the carbapenem resistance of a hypermucoviscous carbapenem-resistant K. pneumoniae strain. The development of the two editing tools will significantly facilitate the genetic engineering of K. pneumoniaeIMPORTANCE Genetics is a key means to study bacterial physiology. However, the highly desirable scarless genetic manipulation is often time-consuming and laborious for the major human pathogen K. pneumoniae We developed a CRISPR-Cas9-mediated genome-editing method and a cytidine base-editing system, enabling rapid, highly efficient, and iterative genome editing in both industrial and clinically isolated K. pneumoniae strains. We applied both tools in dissecting the drug resistance mechanism of a hypermucoviscous carbapenem-resistant K. pneumoniae strain, elucidating that the blaKPC-2 gene was the major factor that contributed to the carbapenem resistance of the hypermucoviscous carbapenem-resistant K. pneumoniae strain. Utilization of the two tools will dramatically accelerate a wide variety of investigations in diverse K. pneumoniae strains and relevant Enterobacteriaceae species, such as gene characterization, drug discovery, and metabolic engineering.

Identification of QTL regions for seedling root traits and their effect on nitrogen use efficiency in wheat (Triticum aestivum L.).

KEY MESSAGE: QTL for a wheat ideotype root system and its plasticity to nitrogen deficiency were characterized. Root system architecture-related traits (RRTs) and their plasticity to nitrogen availability are important for nitrogen acquisition and yield formation in wheat (Triticum aestivum L.). In this study, quantitative trait loci (QTL) analysis was conducted under different nitrogen conditions, using the seedlings of 188 recombinant inbred lines derived from a cross between Kenong 9204 and Jing 411. Fifty-three QTL for seven RRTs and fourteen QTL for the plasticity of these RRTs to nitrogen deficiency were detected. Thirty of these QTL were mapped in nine clusters on chromosomes 2B, 2D, 3A, 3D, 6B, 6D, 7A and 7B. Six of these nine clusters were also colocated with loci for nitrogen use efficiency (NUE)-related traits (NRTs). Among them, three QTL clusters (C2B, C6D and C7B) were highlighted, considering that they individually harbored three stable robust QTL (i.e., QMrl-2B.1, QdRs-6D and QMrl-7B). C2B and C7B stably contributed to the optimal root system, and C6D greatly affected the plasticity of RRTs in response to nitrogen deficiency. However, strong artificial selection was only observed for C7B in 574 derivatives of Kenong 9204. Covariance analysis identified QMrl-7B as the major contributor in C7B that affected the investigated NRTs in mature plants. Phenotypic analysis indicated that thousand kernel weight might represent a "concomitant" above-ground trait of the "hidden" RRTs controlled by C7B, which are used for breeding selection. Dissecting these QTL regions with potential breeding value will ultimately facilitate the selection of donor lines with both high yield and NUE in wheat breeding programs.

NOD2 promotes cell proliferation and inflammatory response by mediating expression of TSLP in human airway smooth muscle cells.

The newly discovered intracytosolic pattern recognition receptor nucleotide-binding oligomerization domain 2 (NOD2) has been studied as an important indicator of T helper 2 (Th2) inflammation, and its effect on regulatory T (Treg) cells is likely to modulate the immune response. In this study, we attempted to study the expression of NOD2 and its impact in human airway smooth muscle cells (HASMC). Quantitative real-time PCR (qRT-PCR) was used to measure the expression level of NOD2 in HASMC and comparisons were made between those from asthmatic and non-asthmatic donors; we found that NOD2 was significantly upregulated in asthma patient tissues and cell lines. In addition, overexpression of NOD2 apparently promotes cell proliferation and migration in HASMC. Gain-of-function in vitro experiments further showed that NOD2 overexpression significantly promotes pro-inflammatory cytokine release in HASMC. Subsequent experimental analysis indicated that thymic stromal lymphopoietin (TSLP) is involved in NOD2-mediated cellular effects in HASMC. Therefore, our results indicate that NOD2 is an asthma-related factor that can promote cell proliferation and inflammatory response by mediated expression of TSLP in HASMC. Taken together, our results indicate that NOD2 could serve as a potential diagnostic biomarker and therapeutic option for human asthma in the near future.

Notch signaling pathway regulates the growth and the expression of inflammatory cytokines in mouse basophils.

Basophils (BAs) are the least common granulocytes of all leukocytes, but they play an important role in orchestrating of chronic allergic inflammation. The Notch signaling pathway is a highly conserved pathway that influences cell lineage decisions and differentiation during various stages of development. However, the relationship between Notch signaling and BA remains to be elucidate. Here, we report that several Notch signaling molecules were found to be expressed in BAs. γ-secretase inhibitor (GSI) treatment increase BAs apoptosis, and suppress BAs proliferation. Furthermore, GSI reduced BAs in the S phase, with a concomitant accumulation in G1 and G2 phases. In addition, GSI also significantly down-regulated mRNA levels of cytokines IL-4, IL-6 and IL-13 induced by A23187, and this effect was dependent on MAPK pathway. Finally, IL-6 inhibition was specifically associated with ERK and IL-13 with JNK. Therefore, Notch signaling regulates BA biological function, at least partially via the modulation of MAPK.